Abstract
Calcineurin (CN), the only Ca2+/calmodulin-activated protein phosphatase and target of immunosuppressant drugs, dephosphorylates substrates within membrane-associated Ca2+ microdomains. CN binds to substrates and regulators via short linear motifs (SLIMs), PxIxIT and LxVP, which have distinct docking sites on CN. PxIxIT binding to CN is Ca2+-independent and affects its distribution, while LxVP associates only with the active enzyme and promotes catalysis. 31 human proteins contain one or more composite LxVPxIxIT motifs, whose functional properties have not been examined. Here we study calcimembrin/C16orf74 (CLMB), a largely uncharacterized intrinsically disordered protein that contains a composite motif which binds to and directs CN to membranes. CLMB overexpression is associated with poor prognoses for several cancers, suggesting that it promotes oncogenesis by shaping CN signaling. We demonstrate that CLMB associates with membranes via N-myristoylation and dynamic S-acylation and is dephosphorylated by CN on threonine 44 (Thr44). The LxVP and PxIxIT portions of the CLMB composite sequence, together with Thr44 phosphorylation, confer high affinity PxIxIT-mediated binding to CN (KD~8.3 nM) via an extended 33LxVPxIxITxx(p)T44 sequence. This binding promotes CLMB-based targeting of CN to membranes, but also protects Thr44 from dephosphorylation. Thus, we propose that CLMB promotes dephosphorylation in multimeric complexes, where one CLMB molecule recruits CN to membranes via PxIxIT binding, allowing other proteins (including CLMB) to engage through their LxVP motifs for dephosphorylation. This unique mechanism makes dephosphorylation sensitive to CLMB:CN ratios and is supported by in vivo and in vitro data including nuclear magnetic resonance (NMR) analyses of CLMB alone and complexed with CN.
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